The causal relationships of granulocytes and melanoma skin cancer: A univariable and multivariable Mendelian randomization study

Abstract Background Increasing evidence has revealed that granulocyte has a critical role in tumorigenesis and progression. In this study, Mendelian randomization (MR) analysis was utilized for estimating the causal association between neutrophil percentage and melanoma skin cancer, eosinophil percentage and melanoma skin cancer, basophil percentage and melanoma skin cancer, respectively. Methods The Genome‐Wide Association Study (GWAS) ids for melanoma skin cancer, neutrophil percentage, eosinophil percentage and basophil percentage were derived from Integrative Epidemiology Unit (IEU) Open GWAS database. The univariable MR (UVMR) analysis was conducted to estimate the risk using MR‐Egger, weighted median, inverse variance weighted (IVW). In addition, sensitivity analysis was conducted to assess the reliability of UVMR results. Finally, the multivariable MR (MVMR) analysis was performed to investigate causality between neutrophil percentage and eosinophil percentage in the presence of both and melanoma skin cancer. Results The UVMR indicated that neutrophil percentage and eosinophil percentage were significantly and causally related to melanoma skin cancer, with neutrophil percentage [p = 0.025, odds ratio (OR) = 1.002] as a risk factor and eosinophil percentage (p = 7.04E‐06, OR = 0.997) as a protective factor. Moreover, MVMR analysis indicated eosinophil percentage remained the protective factor (p = 0.003, OR = 0.998), while the causality of neutrophil percentage and melanoma skin cancer became insignificant (p > 0.05). Conclusion The causal relationships of neutrophil percentage and melanoma skin cancer, eosinophil percentage and melanoma skin cancer were shown by this study, which provided a reference for subsequent research and treatment related to melanoma skin cancer.


INTRODUCTION
Melanoma skin cancer (cutaneous melanoma) represents a highly aggressive and potentially fatal form of cancer stemming from melanocytes. 1 Insights from molecular biology and advanced sequencing techniques have underscored the predominant role of genetic and environmental factors in melanoma skin cancer etiology.Despite constituting only 4% of skin cancer diagnoses, melanoma skin cancer is responsible for a staggering 75% of skin cancer-related deaths. 2,3cording to the GLOBOCAN 2020 database, there were 324 635 new cases of melanoma and 57 043 deaths in 185 countries worldwide in 2020. 4Recent studies suggest that timely detection and appropriate intervention can significantly reduce the mortality rate of melanoma skin cancer.Therefore, the timely detection and prognosis of melanoma emerge as pivotal areas warranting heightened attention and focus.
Numerous potential biomarkers have been utilized to evaluate the prognosis and therapeutic effect of melanoma. 5However, many of these tests are cumbersome, costly, and yield inconsistent results, which has limited their use in clinical practice.[8] Neutrophils, as the most abundant immune cells in the tumor microenvironment, release specific pro-tumoral factors. 9,10Eosinophils are recruited in the peripheral blood of tumor patients, and their levels correlate with patient survival. 11Basophils account for a smaller proportion of the population but can recruit CD8 + T cells to infiltrate, exhibiting potential anti-tumor properties.Previous research has shown that the neutrophil/lymphocyte ratio (NLR) is linked to improved overall survival rates among melanoma patients receiving immune checkpoint inhibitors. 12In addition, Pozorski 13 demonstrated that the neutrophil/eosinophil ratio (NER) can be used as a new prognostic marker in advanced melanoma patients receiving anti-PD-1 therapy.Further investigation is therefore needed into the relationship between granulocyte and melanoma prognosis.
Mendelian randomization (MR) estimates causal effects of exposure and outcome using germline genetic variation as an instrumental variable.Because alleles assort randomly during meiosis and germline genetic variation is fixed at conception, the analysis is immune to conventional confounding and reverse causality in epidemiological observations. 14Genetic evidence for the effect of exposure changes (e.g., granulocyte percentage) on outcome (e.g., melanoma skin cancer prognosis) can therefore be provided by MR studies.To date, no comprehensive MR study of the association between peripheral blood neutrophils/eosinophils/basophils and melanoma prognosis has been reported.
In this study, a univariate and multivariate MR study was performed based on genetic variation data from public databases for melanoma skin cancer and the three exposure factors (eosinophil percentage, neutrophil percentage, and basophil percentage) to investigate the causal effect of granulocyte on melanoma skin cancer, and sensitivity analyses were performed to assess the impact of assumptions on findings and to ensure the robustness of results.

Data source
The

Selection of instrumental variables (IVs)
Three fundamental premises served as the foundation for the MR study: (1) there was a significant correlation between IVs and exposure; (2) the IVs were independent of confounders; (3) the IVs could only influence the outcome through exposure and not through any other channels.

Univariable MR (UVMR) and sensitivity analysis
The input data for UVMR analysis was taken after filtering the IVs.The major MR modes were MR-Egger, 16 simple mode, weighted mode, 17 weighted median, 18 and inverse variance weighted (IVW) regression. 19e most important method was IVW in this study.Odds ratio (OR) was then calculated, with greater than 1 representing a risk factor and less than 1 representing a protective factor.The results were presented using scatter plots, forest plots, and funnel plots.
Sensitivity analyses were conducted to assess the reliability of UVMR results, which mainly consisted of the heterogeneity test, horizontal pleiotropy test and leave-one-out (LOO) analysis.First, Cochran's Q-test was conducted, and there was no heterogeneity when the p-value was greater than 0.05.Second, potential horizontal pleiotropy among IVs was evaluated using MR-Egger test, and when p was greater than 0.05, it indicated no horizontal pleiotropy, meaning that there was no confounding factors in the study.Finally, LOO analysis was conducted to check whether the UVMR results were influenced by any single SNP.

Multivariable MR (MVMR) analysis
To examine the causal relationship between neutrophil percentage and melanin skin cancer, between eosinophil percentage and melanin skin cancer, respectively, at a multivariate level, MVMR analysis was applied.The data processing for MVMR analysis was equivalent to UVMR (p < 5*10 −8 , r 2 = 0.001, kb = 10 000).

Connection of neutrophil percentage, eosinophil percentage, and basophil percentage to melanoma skin cancer
After screening, 262 SNPs for neutrophil percentage, 296 SNPs for eosinophil percentage and 101 SNPs for basophil percentage independent SNPs were obtained as IVs.Table S1 listed the MR results of five different methods.The IVW results showed that neutrophil percentage (p = 0.025) and eosinophil percentage (p = 7.04E-06) were causally associated with melanoma skin cancer.However, melanoma skin cancer was not directly related to basophil percentage (p = 0.706).Observation of OR values revealed that neutrophil percentage (OR = 1.002) was a risk factor and eosinophil percentage (OR = 0.997) was a protective factor for melanoma skin cancer (Figure 1).The scatter plots showed that slope of the line for neutrophil percentage was positive, indicating that increased amount of neutrophil percentage led to increased risk of melanoma skin cancer, and slope of the line for eosinophil percentage showed an opposite trend to neutrophil percentage, indicating that eosinophil percentage was a protective factor for melanoma skin cancer (Figure 2).The SNPs locations in the forest plots were protection on the left and risk on the right.Our findings supported that neutrophil percentage was a risk factor and eosinophil percentage was a protective factor for melanoma skin cancer (Figures S1 and S2).Also, the funnel plots demonstrated that UVMR analysis conformed to Mendel's second law of random grouping (Figure S3).

UVMR analysis results were proven to be reliable through sensitivity analyses
Sensitivity analyses were carried out to determine the reliability of analysis.The p-values of Cochran's Q-test were all less than 0.05, indicating that there was heterogeneity among the selected SNPs (Tables 1   and 2).Although there was heterogeneity, the p-value of the IVW method were all less than 0.05, indicating that the heterogeneity did not significantly affect the results of UVMR analysis, which meant that the results of UVMR analysis were valid.The results of MR-Egger test suggested that there were no horizontal pleiotropy effect among IVs (p > 0.05) in UVMR analysis (Table 3).Meanwhile, the intercept values were all less than 0.01, which meant that there were no confounding factors in our analysis.Furthermore, there was no point of serious bias in the results of LOO analyses, indicating that the UVMR analysis results were reliable (Figures S4 and S5).In conclusion, neu-F I G U R E 1 Forest plot showing the MR results of five different methods between melanoma skin cancer risk and neutrophil (A), eosinophil (B), basophil percentage (C).The IVW results showed that neutrophil percentage (p = 0.025) and eosinophil percentage (p = 7.04E-06) were causally associated with melanoma skin cancer.The OR values for all five methods were in the same direction and all greater than 1, indicating that the percentage of neutrophils is a risk factor for melanoma skin cancer.Conversely, eosinophils have a protective effect.trophil percentage and eosinophil percentage were causally associated with development of melanoma skin cancer, with neutrophil percentage as a risk factor and eosinophil percentage as a protective factor for melanoma skin cancer.

Relationship between neutrophil percentage and eosinophil percentage in the presence of both and melanoma skin cancer by MVMR analysis
After screening, 302 SNPs associated with neutrophil percentage and eosinophil were identified and taken as input data for MVMR analysis.

F I G U R E 2 MR-Egger scatter plot of melanoma skin cancer risk with neutrophil percentage (A) and eosinophil percentage (B).
No changes in the protective or risk properties of two exposure factors were observed after the assessments (Figure 3, Table S2).However, the p-value for neutrophil percentage was greater than 0.05, meaning the causality between neutrophil percentage and melanoma skin can-cer became insignificant.In other words, the influence of eosinophil percentage as a protective factor for melanoma skin cancer was more direct than neutrophil percentage.

DISCUSSION
Melanoma skin cancer stands as the most lethal form of cutaneous malignancy. 20It is imperative to establish a straightforward and efficient method for early diagnosis and prognosis assessment of melanoma. 21Immune checkpoint inhibitor (ICI)-based therapies (especially those targeting anti-cytotoxic T-lymphocyte-associated antigen-4 (anti-ctla-4) and anti-programmed death-1 (anti-pd-1) antibodies) have dramatically improved overall survival in melanoma in recent years, 22 but unfortunately, there are still no validated biomarkers to indicate the therapeutic prognosis of ICL.Given the simplicity and convenience of peripheral blood tests, coupled with the pivotal role of granulocytes in tumorigenesis and progression, we aimed to investigate the significance of peripheral blood granulocyte percentage in the prognostic determination of melanoma. 23udying correlations can be challenging due to the complex and time-consuming design of prospective studies.Additionally, changes in granulocytes in peripheral blood are associated with various factors, which can lead to biased epidemiological studies.Therefore, providing strong evidence can be difficult.It is important to acknowledge the limitations of traditional epidemiological studies.Due to the potential for bias in experimental designs and clinical studies, the causal relationship between exposure and disease can often be influenced by confounding factors.To address this issue, MR analysis offers a new approach for assessing the causal relationship between exposure and disease by using genetic variation. 24Genetic variants can be used as a randomized controlled trial (RCT) because they are assigned to individuals before any exposure or outcome occurs, and this assignment is randomized.This reduces the occurrence of reverse causation and potential confounders, making the results similar to those of an RCT. 24,25evious studies have employed MR to investigate the crucial role of inflammation-related factors in the progression of certain skin Forest plot for MVMR analysis of melanoma skin cancer with neutrophil percentage and eosinophil percentage.The p-value for eosinophil percentage remained below 0.05, indicating a more direct impact on melanoma skin cancer.
diseases and malignant tumors. 26,27This study presents the first MR analysis to investigate the causal relationship between the percentage of neutrophils and eosinophils in peripheral blood and prognosis in melanoma.It is the largest and most comprehensive study of its kind to date.
Our study found a significant causal effect of neutrophil percentage on melanoma skin cancer and that neutrophil percentage was a risk factor.It is well known that lymphocytes have a well-established anti-tumor role, and their immune surveillance and destruction of neoplastic tumors are essential.With respect to neutrophils, it can inhibit lymphocyte proliferation and induce lymphocyte apoptosis.
It is widely accepted that neutrophils are positively correlated with tumor progression, 9,28,29 which is consistent with our findings.Neutrophils expressing PD-L1 can induce T-cell apoptosis by binding to lymphocyte PD-1. 30In addition, neutrophils can directly reduce lymphocyte proliferation by producing reactive oxygen species and arginase-1.Furthermore, neutrophil extracellular traps (NETs) have been shown to enhance tumor cell adhesion and promote extracellular matrix degradation, thereby facilitating tumor metastasis. 31,32Based on this, numerous studies have investigated the reliability of the neutrophil-to-lymphocyte ratio (NLR) as a means of determining the prognosis of melanoma. 33A meta-analysis of 12 studies and 3207 patients 34 with stage I-IV melanoma revealed that a high NLR is independently associated with higher melanoma-specific mortality, leading to significantly worse overall survival.However, Wade et al. 35 conducted a multicenter cohort study with a total of 1351 patients (66% stage I) and found that high NLR was positively associated with better overall survival and disease-free survival.In the early stages of melanoma, neutrophils have been found to be more anti-tumor.
Further studies have confirmed that the main regulator of the pro-or anti-tumorigenic neutrophil phenotype is Notch1, which is elevated to a greater extent in advanced melanoma. 36rthermore, there was a significant causal relationship between eosinophil percentage and melanoma skin cancer in our study, and eosinophil percentage was a protective factor for the risk of melanoma skin cancer. 37,38Research on the relationship between eosinophils and melanoma prognosis is limited.a predictive biomarker for the anti-PD-1 treatment of advanced melanoma. 39,40at is more, after conducting a MVMR analysis, it was found that the p-value of neutrophil percentage was greater than 0.05, indicating that the causal relationship between neutrophil percentage and melanoma skin cancer became insignificant.Therefore, the effect of eosinophil percentage as a protective factor for melanoma skin cancer is more direct than that of neutrophil percentage.This partially supports prior research indicating that neutrophils play distinct roles during various stages of melanoma development.They may exhibit anti-tumor properties during the early stages of tumor growth, but overall, they still exhibit tumorigenic properties.In contrast, eosinophils demonstrate more direct anti-tumor properties, and a higher percentage of eosinophils suggests a better prognosis for melanoma.
There are limitations to this study.Firstly, the data analyzed were mainly from populations of European origin.Therefore, a larger sample of multiethnic groups with more relevant SNPs may have contributed to more reliable conclusions.Secondly, due to the limitations of the sample and clinical data, subgroup analyses were not possible.For example, the role of neutrophils at different stages of tumor development remains unclear.This prevented us from using MR to gain further insight into the study for clinical purposes.

CONCLUSION
Our study have shown a potential causal relationship between the percentage of eosinophils and neutrophils with melanoma skin cancer using UVMR.However, the MVMR analysis did not find a significant causal relationship between neutrophil percentage and melanoma skin cancer.On the other hand, eosinophil percentage remained a direct protective factor against melanoma.The data suggests that a decrease in eosinophil percentage is directly associated with the development of melanoma skin cancer, while altered neutrophil percentage may not be directly associated with it.Therefore, it is important to monitor the eosinophil percentage of patients and consider it in the prognosis of melanoma skin cancer.Early targeted treatment should be considered based on this information.
The heterogeneity of neutrophil instrumental variables.indicating that the heterogeneity between the neutrophil percentage and melanoma skin cancer datasets did not significantly affect the analysis results, which meant the analyses were valid.Horizontal pleiotropy assessed of the association between neutrophil/eosinophil and melanoma skin cancer.
TA B L E 1 TA B L E 2The heterogeneity of eosinophil instrumental variables.Note:*p<0.05,indicating that the heterogeneity between the eosinophil percentage and melanoma skin cancer datasets did not significantly affect the analysis results, which meant the analyses were valid.TA B L E 3